Project Summary The cardiac action potential is primarily generated by sodium and calcium channels, which depolarize the membrane potential, and by potassium channels that repolarize the membrane potential and terminate the action potential. One of the major cardiac potassium currents is the slowly activating potassium current IKs that contribute to the action potential termination. Over 300 different inherited mutations have been found in IKs channels that cause cardiac arrhythmias in patients. IKs channels regulate the length of the cardiac contraction and mutations that decreases the activity of IKs channels result in a prolongation of the cardiac contraction, leading to Long QT Syndrome. In turn, Long QT syndrome is a risk factor for ventricular fibrillation and sudden cardiac death. We have recently identified a family of compounds that act on IKs channels and are antiarrhythmic when applied to cardiomyocytes. We will here test whether these compounds are anti- arrhythmic in human cardiomyocytes using whole-cell patch clamp and a novel pseudo-ECG in-a-dish measurements on a monolayer of human cardiomyocytes. We will also test the effect of these compounds on whole animal heart and in whole animals, in order to develop an anti-arrhythmic drug that can be tested in future clinical trial. We will also test variants of these compounds on heterologously expressed IKs channels using two-electrode voltage clamp, to determine the important structure of these compounds for their effects on IKs channels. We will also make mutations of IKS channels to determine the binding site of these compounds. Finally, we will test the efficacy of these compounds to reverse different defects in IKs channels caused by different types of Long QT syndrome mutations. This will be tested both in heterologous systems and in human cardiomyocytes. The anticipated results of these experiments will provide proof-of-concept that this family of compounds acts anti-arrhythmic and will provide preliminary animal model data to start clinical trials of these compounds. We anticipate that this development of new anti-arrhythmic drugs will lead to better treatments of cardiac arrhythmias and the prevention of sudden cardiac deaths.